Department of Computer Science, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
Department of Life Science Frontiers, Center for iPS Cell Research and Application, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan.
ACS Nano. 2024 Jun 18;18(24):15477-15486. doi: 10.1021/acsnano.3c12161. Epub 2024 Jun 3.
DNA droplets, artificial liquid-like condensates of well-engineered DNA sequences, allow the critical aspects of phase-separated biological condensates to be harnessed programmably, such as molecular sensing and phase-state regulation. In contrast, their RNA-based counterparts remain less explored despite more diverse molecular structures and functions ranging from DNA-like to protein-like features. Here, we design and demonstrate computational RNA droplets capable of two-input AND logic operations. We use a multibranched RNA nanostructure as a building block comprising multiple single-stranded RNAs. Its branches engaged in RNA-specific kissing-loop (KL) interaction enables the self-assembly into a network-like microstructure. Upon two inputs of target miRNAs, the nanostructure is programmed to break up into lower-valency structures that are interconnected in a chain-like manner. We optimize KL sequences adapted from viral sequences by numerically and experimentally studying the base-wise adjustability of the interaction strength. Only upon receiving cognate microRNAs, RNA droplets selectively show a drastic phase-state change from liquid to dispersed states due to dismantling of the network-like microstructure. This demonstration strongly suggests that the multistranded motif design offers a flexible means to bottom-up programming of condensate phase behavior. Unlike submicroscopic RNA-based logic operators, the macroscopic phase change provides a naked-eye-distinguishable readout of molecular sensing. Our computational RNA droplets can be applied to in situ programmable assembly of computational biomolecular devices and artificial cells from transcriptionally derived RNA within biological/artificial cells.
DNA 液滴是经过精心设计的 DNA 序列的人工类液相凝聚物,可实现相分离生物凝聚物的关键方面的可编程利用,如分子传感和相态调节。相比之下,尽管 RNA 具有更多样化的分子结构和功能,从类似 DNA 的特征到类似蛋白质的特征,但它们的 RNA 对应物的研究仍较少。在这里,我们设计并展示了能够进行双输入 AND 逻辑运算的计算型 RNA 液滴。我们使用多分支 RNA 纳米结构作为构建块,该结构由多个单链 RNA 组成。其分支参与 RNA 特异性亲吻环 (KL) 相互作用,使自组装成网络状微观结构。当接收到两个目标 miRNA 的输入时,纳米结构被编程为分裂成低价结构,这些结构以链式方式相互连接。我们通过数值和实验研究相互作用强度的碱基可调性,优化了源自病毒序列的 KL 序列。只有在接收到同源 miRNA 时,RNA 液滴才会由于网络状微观结构的解体而选择性地从液相变为分散状态,从而发生剧烈的相态变化。这一演示强烈表明,多股基序设计为凝聚相行为的自下而上编程提供了一种灵活的手段。与亚微观 RNA 逻辑运算器不同,宏观相变化提供了分子传感的肉眼可区分的读出。我们的计算型 RNA 液滴可应用于转录衍生的 RNA 在生物/人工细胞内的计算型生物分子器件和人工细胞的原位可编程组装。